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ABSTRACT

Study Objectives:

Narcolepsy is caused by a selective loss of hypocretin neurons and is associated with obesity. Ghrelin and leptin interact with hypocretin neurons to influence energy homeostasis. Here, we evaluated whether human hypocretin deficiency, or the narcolepsy therapeutic agent sodium oxybate, alter the levels of these hormones.

Conclusions:

The increased BMI of narcolepsy patients is unlikely to be mediated by hypocretin deficiency-mediated alterations in total ghrelin or leptin levels. Thus, the effects of these hormones on hypocretin neurons may be mainly unidirectional. Although sodium oxybate may influence body weight, the underlying mechanism is unlikely to involve changes in total ghrelin or leptin secretion.

The hypocretin system, also known as the orexin system, is of major importance in the regulation of sleep and sustained wakefulness. Moreover, hypocretin neurons are responsive to metabolites and hormones helping to translate signals of metabolic state into adaptive levels of activity and consciousness.1,2 Hypocretin deficiency leads to narcolepsy, a sleepwake disorder characterized by excessive daytime sleepiness, cataplexy, and disrupted nocturnal sleep. Obesity is associated with the disorder,3 yet the cause of the increased body weight has been challenging to discern due to inconsistent findings on the hormonal and metabolic characteristics of this population.4–9 However, altered ingestive behavior has been consistently observed in these patients,7,10–12 suggesting hypocretin deficiency may dysregulate feeding behavior, and possibly energy homeostasis.

BRIEF SUMMARY

Current knowledge/Study Rationale: Potential hormonal disturbances in response to hypocretin deficiency may provide explanation for the weight and ingestive behavior phenotype of this population. The relationship of both ghrelin and leptin with hypocretin has been shown to be of importance. Hypocretin neurons directly sense ghrelin and leptin but it is unknown if these connections are uni-or-bidirectional. Therefore, the study of hypocretin-deficient narcoleptic patients provides a unique opportunity to further explore the nature of these relationships in this population of patients in the chronic phase of this disorder.

Study Impact: No significant alteration in total ghrelin or leptin levels were seen in our sample of hypocretin-deficient narcoleptic subjects. Therefore, we found no evidence to support a bidirectional, integrated feedback loop for total ghrelin or leptin levels and hypocretin. To our knowledge, total ghrelin levels have not previously been reported in human narcolepsy and our findings will help others to better understand the hormonal phenotype of this population. Our findings are in support of the notion that the leptin level is not altered in this population, although we did see a significant increase in leptin pulse frequency in subjects compared to controls; the clinical significance of this finding is unknown. Our findings suggest the propensity for obesity and the altered ingestive behavior of narcolepsy are unlikely to be mediated by hypocretin-deficiency derived alterations to these hormone levels.

Ghrelin is a peptide hormone mainly produced by endocrine cells in the stomach and gastrointestinal tract, and is an important endogenous regulator of energy balance and growth hormone (GH) secretion.13 Its expression is complex14 and influenced by sympathetic nervous system activity.15 Across the wake period, plasma concentrations wax and wane episodically, providing an appetite-stimulating signal to the brain.16 During sleep, ghrelin levels rise sharply in the early part of the night and decrease gradually toward morning. During sleep deprivation, however, levels gradually rise toward a plateau in the morning.17 Hypocretin neurons directly sense and are excited by ghrelin, and an interaction between these two systems has been shown to be involved in ingestive behavior.18 A study by Toshinai et al.19 first identified this connection. In that study, ghrelin-induced feeding was attenuated in rats pretreated with antihypocretin-1 IgG and antihypocretin-2 IgG, and suppressed in hypocretin knockout mice. Later, it was demonstrated that ghrelin plays a key role in the rewarding aspects of eating,20 but it requires the presence of intact hypocretin signaling to impart this effect.

Leptin is another peptide hormone involved in energy homeostasis, the dominant role of which is to signal energy deficiency to the brain.21 It is an adipokine produced primarily by subcutaneous white adipose tissue, and its expression is stimulated by various hormones, sympathetic outflow, energy intake, and output.21,22 Under normal conditions, blood levels display circadian variation as levels rise across the day and peak in the middle of the night.23 During sleep deprivation, blood leptin levels show a reduced and flattened profile.24 Receptors for leptin are found on hypocretin cells,25 and leptin can directly inhibit the expression of isolated hypocretin neurons.18 Indirectly, leptin can affect the activity of hypocretin cells via energy-regulating neurons in the arcuate nucleus of the hypothalamus.26 Conversely, because the hypocretin system greatly influences autonomic control,1 it is plausible that hypocretin deficiency may alter leptin expression via inhibited sympathetic activity. Indeed, obese hypocretin deficient mice have lowered sympathetic vasoconstrictor outflow, while greater heart rate variability has been observed in hypocretin deficient narcolepsy patients.27,28 Thus, leptin and hypocretin may interact to affect levels of physical activity and wakefulness in response to energy needs, and the loss of hypocretin neurons may dysregulate leptin expression and signaling.

While ghrelin levels have not been previously reported in hypocretin-deficient narcoleptic patients, abnormal leptin levels have been observed.4,5 It is unknown if the associations between hypocretin and total ghrelin or leptin are uni-or-bidirectional. Because hypocretin influences sympathetic outflow and sympathetic nervous system activity affects the expression of both leptin and ghrelin, hypocretin deficiency may lead to altered levels of these hormones. This study of hypocretin-deficient narcoleptic patients provides a unique opportunity to further explore the nature of these relationships. We hypothesized that both total ghrelin and leptin levels would be abnormal in hypocretin-deficient narcolepsy patients, which might help explain the increased BMI and abnormal ingestive behavior seen in this population.5,7,8,29–31

Additionally, we explored if the narcolepsy therapeutic agent, sodium oxybate, has an effect on these hormones. In a narcolepsy population, sodium oxybate improves disrupted nocturnal sleep, impaired wakefulness, and cataplexy, and promotes weight loss.32,33 Like ghrelin, sodium oxybate administration also stimulates GH release.34 We hypothesized that its administration would alter total ghrelin levels, the effect of which might be involved in its GH-promoting effects.

Here, we investigate whether total blood ghrelin or leptin levels are altered in hypocretin-deficient narcoleptic patients compared to controls, and whether total ghrelin or leptin levels are influenced by sodium oxybate.

MATERIALS AND METHODS

Subjects

We included 8 medicationfree, male hypocretin-deficient narcolepsy with cataplexy patients and 8 healthy male controls, matched for age, BMI, and body fat percentage. Hypocretin measurement was performed according to international standards.35 Body fat percentage was measured with bioelectrical impedance analysis (Bodystat, Douglas, Isle of Man, UK). Two patients were drug naive, one patient was tapered from antidepressants ≥ 2 weeks prior to the study, and 2 patients had prior history with sodium oxybate therapy; however, no subject took sodium oxybate within 20 days of study initiation. The other patients did not take any medication for at least several months prior to beginning the study.

Subjects were eligible for participation after exclusion of chronic conditions, with particular attention to the absence of sleep disorders in control subjects, hypertension, pituitary disease, and weight change (> 3 kg weight gain or loss within the last 3 months) as assessed by structured clinical interview. None of the participants had previously undergone gastrectomy. Written informed consent was obtained from all subjects. The study was approved by the ethics committee of the Leiden University Medical Center.

Clinical Protocol

All subjects were admitted to the Clinical Research Center for 24-h blood sampling before and after 5 days of sodium oxybate administration. A cannula was inserted into an antecubital vein ≥ 45 min before the start of blood sampling at 12:00. Blood samples were collected with S-monovetten (Sarstedt, Etten-Leur, The Netherlands) from a 3-way stopcock that was attached to a 0.9% NaCl and heparin (1 U/mL) infusion (750 mL/24 h) to keep the cannula from clotting. For total ghrelin measurements, blood was collected in EDTA tubes at 60-min intervals, and these tubes were immediately put on ice. Ghrelin samples were acidified with 50 μL of 1 N HCL. Within 5 min of sampling, tubes were centrifuged at 1,250 g at 4°C for 20 minutes. For leptin measurements, blood was collected at 20-min intervals. After clotting, the blood was centrifuged within 30 min of sampling (20 min, 1,250 g, 4°C). Serum was then stored at -80°C until hormonal assays. Three standardized meals were served at 08:30, 13:00, and 18:00 (Nutridrink, 1.5 kcal/mL, 2,100 kcal/d; macronutrient composition per 100 mL: protein, 6 g; fat, 5.8 g; carbohydrate, 18.4 g; Nutricia, Zoetermeer, The Netherlands). Subjects were asked to complete each meal provided. Foodinduced suppression of total ghrelin release was defined as the ratio between total ghrelin levels one hour postprandially to the levels immediately before the meal (lunch and dinner) or 30 min postprandially to 30 min before the meal (breakfast). Subjects remained sedentary except for bathroom visits. In both study occasions, lights were switched off (dark period) at 23:00 and then switched on at 07:30.

Sodium Oxybate

In the drug-intervention study occasion, sodium oxybate was administered in a total nightly dose of 6 grams/night for 5 consecutive nights in both the narcoleptic patients and the controls. Each night, 3 grams of sodium oxybate were administered orally at 23:00 and 03:00. Lights were turned off after ingestion of the first dose.

Sleep Recordings

During the 24-h sampling periods, polysomnographic recordings were performed using an ambulant EEG-recording system (Embletta X100, Embla) and a standard EEG/EMG montage to allow sleep scoring according to AASM criteria. Using a markertool, the start of the sampling protocol was registered to synchronize sleeprecordings with hormone measurements. Sleep recordings were scored by an experienced technician, blinded for the subject under study.

Assays

Plasma total ghrelin and leptin levels were measured by radioimmunoassay (LINCO Research, St. Charles, MO, USA) with a detection limit of 93 pg/mL, and an interassay variation ranging from 14.7% to 17.8% for total ghrelin and a detection limit of 0.5 μg/L and an interassay variation ranging from 3.0% to 5.1% for leptin. Samples from each patient and matched control were handled in the same run.

Deconvolution Analysis

Leptin concentration time series were analyzed via a recently developed automated deconvolution method, empirically validated using hypothalamopituitary sampling and simulated pulsatile time series.36 The Matlabbased algorithm first detrends the data and normalizes concentrations to the unit interval [0, 1]. Second, the program creates multiple successive potential pulsetime sets, each containing one fewer burst via a smoothing process (a nonlinear adaptation of the heatdiffusion equation). Third, a maximum-likelihood expectation estimation method computes all secretion and elimination parameters simultaneously conditional on each of the multiple candidate pulsetime sets. The fast halflife was represented as 3.4 min constituting 19% of the decay amplitude. The slow halflife was estimated as an unknown variable between 6 and 70 min. Here we present only results for pulse frequency (pulses per 24 h), basal secretion, pulsatile secretion, and total secretion per 24 h, all expressed as μg per liter distribution volume.

Data Analysis and Statistics

Results are expressed as mean ± standard deviation (SD). Unpaired ttests were used to assess differences in means between the 2 groups, while paired ttests were applied to assess changes in means within each group. All tests were 2-tailed, and significance level was set at p < 0.05. Statistical analyses were performed using SPSS for Windows (release 17.0, SPSS, Inc., Chicago, IL).

RESULTS

Subjects

Patients and controls did not differ with respect to age, BMI, waisttohip ratio, and body fat percentage (Table 1). Sodium oxybate was well tolerated by all participants. Apart from mild drowsiness, no other side effects were reported during the study.

Demographics, body composition, baseline parameters

Table 1

Sleep and Wakefulness Differences

When compared to controls, during baseline conditions and after sodium oxybate administration, narcolepsy patients spent significantly less time awake across a 24 h period, and during the day (defined as the lightson period between 07:30-23:00) they spent less time awake and more time in slow wave sleep ([SWS] p = 0.004 and p = 0.005, respectively; Table 2).

Baseline Total Ghrelin Levels

Mean 24-h total ghrelin levels at baseline were virtually identical between narcolepsy patients and controls (p = 0.873; Figure 1A). Mean total ghrelin levels were also not different between the 2 groups when the analyses were restricted to the dark period (p = 0.973). In fact, at no single timepoint an intergroup difference could be detected (all p ≥ 0.232). Food induced suppression of total ghrelin concentration (expressed as the ratio between postprandial to preprandial total ghrelin concentration) was similar in the 2 groups (lunch: p = 0.413, dinner: p = 0.301, breakfast: p = 0.437, and mean postprandial total ghrelin levels averaged over the 3 occasions [p = 0.540]) (Table 3).

Plasma ghrelin concentrations and deconvolution of leptin levels before and after administration of sodium oxybate in both narcoleptic patients and controls

Table 3

Plasma ghrelin concentrations and deconvolution of leptin levels before and after administration of sodium oxybate in both narcoleptic patients and controls

Mean 24-h ghrelin levels in narcolepsy patients and matched controls

The diurnal plasma ghrelin levels, as well as food induced suppression of ghrelin release were not significantly different between narcolepsy patients and matched controls, either during basal conditions (A) or after 5 days of sodium oxybate administration (B). Hourly blood sampling started at noon and continued for 24 hours. The black bar on the abscissa indicates the dark period (23:00-07:30). The gray arrows indicate the timings of the lunch, dinner, and breakfast at 13:00, 18:00, and 08:30, respectively. The black arrows indicate the timings of sodium oxybate administrations during the second study occasion at 23:00 and 03:00. Error bars show the means ± standard deviation.

Figure 1

Mean 24-h ghrelin levels in narcolepsy patients and matched controlsThe diurnal plasma ghrelin levels, as well as food induced suppression of ghrelin release were not significantly different between narcolepsy patients and matched controls, either during basal conditions (A) or after 5 days of sodium oxybate administration (B). Hourly blood sampling...

Effect of Sodium Oxybate on Total Ghrelin Levels

Twentyfour hour mean total ghrelin levels during sodium oxybate treatment were not different between narcolepsy patients and controls (p = 0.642; Figure 1B). Similar to baseline, mean total ghrelin levels during the dark period did not differ between the 2 groups (p = 0.449), and at no single timepoint a difference could be detected between groups (all p ≥ 0.05). Postprandial total ghrelin suppression, as defined above, was also similar between the 2 groups after sodium oxybate administration: lunch (p = 0.920), dinner (p = 0.261), and breakfast (p = 0.880); mean postprandial total ghrelin levels averaged over the 3 occasions (p = 0.428) (Table 3). The average change in 24-h total ghrelin levels between the second and first occasion amounted to -15 ± 72 pg/mL in narcolepsy patients and -63 ± 87 pg/mL in controls, but was not significantly different from zero in either group (paired ttests: p = 0.56 and p = 0.078, respectively).

Baseline Leptin Levels

Mean 24-h total leptin levels at baseline were not significantly different between narcolepsy patients and controls (p = 0.18; Figure 2A). Mean pulse frequency was different between the 2 groups (p = 0.04), but mean 24-h basal and pulsatile secretion levels were not different (p = 0.96; p = 0.11, respectively; Table 3).

Mean 24-h plasma leptin concentration

Mean 24-h plasma leptin concentration ± SD, before (A) and during sodium oxybate administration (B) in narcolepsy patients and matched controls. The black horizontal bar on the abscissa indicates the lights off period. The gray arrows indicate the timing of meals and the black arrows indicate timing of sodium oxybate administration (B).

Figure 2

Mean 24-h plasma leptin concentrationMean 24-h plasma leptin concentration ± SD, before (A) and during sodium oxybate administration (B) in narcolepsy patients and matched controls. The black horizontal bar on the abscissa indicates the lights off period. The gray arrows indicate the timing of meals and the black arrows indicate...

DISCUSSION

We found no differences in mean 24-h total plasma ghrelin levels or foodinduced suppression of ghrelin concentrations between narcolepsy patients and controls, nor any influence of 5 days of sodium oxybate administration in both groups. In view of the capacity of ghrelin to stimulate growth hormone secretion, it is worth noting that a report from this same research protocol showed no differences in mean hourly GH levels between patients and controls, supporting our conclusion that total ghrelin levels are not altered with hypocretin deficiency.37

Despite the excitatory influence of ghrelin on hypocretin neurons and the interaction of the ghrelin-hypocretin systems to influence food reinforcement, our finding did not show the total ghrelin level to be influenced by hypocretin deficiency, suggesting a unidirectional relationship. These findings also suggest that disturbed ingestive behavior is unlikely mediated by an altered total ghrelin level in narcolepsy patients. Notably, we measured total ghrelin levels and not the biologically active, octanoylatedghrelin fraction. While there is a high correlation between the total and octanoylated fraction ghrelin level,38 it remains possible that the active fraction may be altered in this population.

In contrast to earlier reports,4,5 more recent, larger, controlled studies have not demonstrated an abnormal leptin level in humans with hypocretin deficiency.6,39 Similar to the recent research on this subject, we found that the mean 24-h total leptin level, and basal and pulsatile secretion levels were not significantly different between narcolepsy patients and controls. The mean leptin pulse frequency was slightly but significantly higher in narcolepsy patients in both conditions, but the clinical relevance of this finding is unclear. Because sleep disruption and insulin resistance40 have been shown to affect leptin levels, it is plausible that previous investigations showing decreased leptin in narcolepsy may have resulted from a study sample of narcoleptic patients with relatively poor sleep or a difference in insulin sensitivity compared to the control group.

There were several limitations to the study. The small number of patients and controls raise the possibility of a type II statistical error. However, the intergroup differences were very small; therefore, a large sample size would be needed to detect a difference if present. As with many chronic diseases, compensatory mechanisms are likely involved in narcolepsy as the condition progresses from onset into the chronic stage. Studying narcoleptic patients only during the chronic stage challenges the interpretation that loss of hypocretin cells in the hypothalamus does not alter leptin and ghrelin levels since compensatory adaptation may have already taken place. However, alterations in appetite and weight regulation remain present and clinically relevant in the chronic stage of the disease, and therefore our findings remain relevant despite putative compensations. Additionally, since sleepwake state instability is intrinsic to hypocretin deficiency, standardizing research parameters such as study environment, meal timing and composition, and predefined bedtimes may have created a setting not representative of reallife conditions for these patients. Therefore, although we did not find alterations in total ghrelin and leptin concentrations in this controlled and standardized environment, it remains possible that the release of these hormones is affected by the altered sleep, wake, and eating patterns described in this population.

As expected, in both groups nighttime administration of sodium oxybate increased SWS and reduced awakenings, and the narcoleptic patient group showed a trend towards increased wakefulness the following day. As demonstrated in other studies, acute sodium oxybate administration corresponds with a significant increase in GH release.34,37 However, we found no evidence that the GH-elevating effect is mediated through an influence on total ghrelin secretion. Various treatment effects of sodium oxybate exhibit discrete temporal dynamics with some effects occurring acutely and other effects taking place only after chronic exposure. Although the difference in total ghrelin levels between patients and controls after sodium oxybate administration was not significant, it is possible that significant differences would be seen with higher doses, prolonged periods of nightly administration, or in a larger group of subjects. Lastly, we did not see an effect of sodium oxybate on the leptin level, and to our knowledge, an interaction between this drug and hormone has not been reported elsewhere.

Therefore, mechanisms underlying increased BMI and altered ingestive behavior in narcolepsy and the effects of sodium oxybate administration on GH release and weight loss are unlikely to involve changes in total plasma ghrelin or leptin concentrations. Future investigations should further evaluate if the sleepwake instability intrinsic to hypocretin-deficient narcolepsy promotes ingestive and activity patterns that promote positive energy balance.

DISCLOSURE STATEMENT

This study was supported by an unrestricted grant from UCB Europe. Mr. Pardi was previously employed with Jazz Pharmaceuticals and has consulted with UCB Europe. Dr. Overeem has consulted for and received honoraria as a speaker from UCB Europe. Dr. Lammers is member of the international advisory board on narcolepsy for UCB and has received honoraria as a speaker from UCB. The other authors have indicated no financial conflicts of interest.